PROJECT ABSTRACT It is now understood that in their natural environments, bacteria primarily exist in multicellular, surface-bound communities called biofilms. Biofilms cause major problems in medicine as they are inherently resistant to antibiotics and cause chronic infections; in industry, biofilms foul surfaces and clog filtration devices. Cells in biofilms display striking differences from planktonic cells, such as extracellular matrix production, a 1,000-fold increase in tolerance to antibiotics, and unique gene expression patterns that are specific to particular locations within the biofilm. Because biofilms are three dimensional, heterogeneous, and rearrange over time, investigations have been limited to optical studies of biofilm formation when only few cells are present or to gross characterization of the entire structure. We recently made a research breakthrough: We resolved the individual cells in living, growing biofilms up to a depth of 30 microns, using customized spinning-disk confocal microscopy, fluorescent reporters, and automated cell-segmentation software. This is the first time anyone has peered into a biofilm, to watch it develop, cell by cell, in the presence of flow, under conditions that model environmental, medical, and industrial systems. Thus, we are in a position to use three-dimensional imaging, combined with key technological advancements they propose to make in photo-activation and optogenetics, to characterize biofilms from the gene to the genome and from the cell to the collective. Central questions to be addressed for the first time include how do quorum sensing and genome-wide expression profiles vary in space and time within growing biofilms? Experimental design and interpretation of measurements will be guided by biophysical modeling. We will launch the studies with the human pathogen Vibrio cholerae, known for rapid but transient biofilm formation. Specifically, we will pioneer a comprehensive examination of biofilm formation, development, and signal transduction from the single-cell to multi-cell levels and in realistic environments that mimic the spatial, temporal, and physical constraints found in nature. The interdisciplinary work will lead to understanding of gene regulation, cell-to-cell communication, and the spatial and temporal organization of biofilms, which in turn, dictate the large- scale features and ecological fitness of these multicellular systems. The proposal is unusually interdisciplinary: it teams Bassler, a microbiologist who is a leader in quorum sensing and biofilms, with Stone, an engineer whose focus is imaging, fluid dynamics, and the modeling of transport processes, and Wingreen, a theoretical biophysicist who models bacterial signaling circuits and biofilm development. The approach of direct imaging, beyond connecting genetics to biophysics, promises new insights relevant to understanding and manipulating biofilms with the goal of improving human health.